Oxidants like hypochlorous acid, the active ingredient of household bleach, are powerful bactericides. They are produced by higher eukaryotic organisms to fight infections and limit bacterial colonization. In 1999, we identified the redox-regulated molecular chaperone Hsp33, which is specialized to protect bacteria against these oxidants. In contrast to many proteins that are damaged by oxidants like hypochlorous acid, Hsp33 is rapidly activated. Hsp33 uses a combination of regulatory features to specifically sense these oxidants and respond with extensive conformational rearrangements that lead to the activation of its chaperone function. This activation of Hsp33 apparently compensates for other major bacterial chaperone systems, like the DnaK system, which are rapidly inactivated under these stress conditions. We will now investigate the mechanism of Hsp33's functional regulation. Our studies will address fundamental questions in redox biology, chaperone function and protein folding. Using structural tools, we will elucidate how Hsp33, as a redox-regulated protein, utilizes oxidative stress-mediated domain unfolding as mechanism of activation. We will visualize the precise conformational changes that determine Hsp33's activity state and use site-specific mutagenesis to locate the high affinity substrate-binding site in Hsp33. We will conduct mechanistic studies to obtain insights about how Hsp33, as ATP-independent chaperone holdase, utilizes the ATP-dependent chaperone system DnaK to control its substrate protein release upon return to non stress conditions. Finally, we will determine the fate of Hsp33's substrate proteins upon their release from Hsp33 to obtain a picture about how proteins recover from oxidative stress-induced damage. PUBLIC HEALTH RELEVANCE: Oxidants like bleach are used as disinfectants not only by millions of people around the world, but also by human cells to defend themselves against invading bacteria. We will investigate how a bacterial protein called Hsp33, which is specifically activated by bleach, works to help bacteria survive this oxidative attack. This work may lead to the development of new antibiotics that specifically target these bacterial defense systems.